Hybridization of potato spindle tuber viroid to cellular DNA of - NCBI

Proc. Natd. Acad. Sci. USA Vol. 73, No. 7, pp. 2453-2457, July 1976

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Cell Biology

Hybridization of potato spindle tuber viroid to cellular DNA of normal plants* (viroid/molecular hybridization/Solanaceae/host origin)

A. HADIDIO, D. M. JONESf, D. H. GILLESPIEt, F. WONG-STAALt, AND T. 0. DIENERt Virology Laboratory, Plant Protection Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705; and Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryand 20014

t Plant

Communicated by Heinz Fraenkel-Conrat, May 10, 1976

ABSTRACT Molecular hybridization experiments of 125Ilabeled potato spindle tuber viroid (PSTV) with DNA from uninfected or PSTV-infected tomato plants showed that infrequent DNA sequences complementary to PSTV exist in both uninfected and infected cells. DNA titration experiments revealed that at least 60% of PSTV is represented by sequences in DNA of several normal solanaceous host species. Phylogenetically diverse plants contain sequences related to less of the PSTV. PSTV-infected tomato or Gynura aurantiaca plants did not possess new PSTV sequences at detectable levels. These results support the hypothesis that PSTV may have originated from genes in normal solanaceous plants.

Potato spindle tuber viroid (PSTV), the causal agent of potato spindle tuber disease, is a small RNA of about 8 X 104 daltons (1, 2) that lacks poly(A) or poly(C) sequences in its structures. This free RNA is able to systemically infect plants of several families, especially species of the Solanaceae (3-5); and it appears to replicate autonomously in susceptible cells (6). PSTV is of sufficient chain length to code for a polypeptide of about 1 X 104 daltons; however, in vitro studies indicated that PSTV is not translated in several cell-free systems which synthesize protein (7). Whether PSTV replication involves a DNA intermediate is not known, but recent evidence showed that PSTV replication in tomato leaf strips (8) or tomato nuclei (9) is inhibited in the presence of actinomycin D, a result which suggests the involvement of DNA. Molecular hybridization experiments with PSTV and plant cellular DNA or RNA can be used to determine whether sequences complementary to PSTV occur in the DNA or RNA of susceptible plant species or whether such sequences appear as a consequence of infection with PSTV. In this paper, we report that PSTV hybridizes with cellular DNA of several uninfected host species (Solanaceae) and that infection with PSTV has no detectable effect on the hybridization pattern. PSTV exhibits less complementarity with DNA of nonsolanaceous plants and PSTV sequences are not introduced into one of these plants (Gynura aurantiaca) upon infection. Our results suggest a cellular origin for PSTV and indicate that the requirement of DNA synthesis for PSTV replication does not involve synthesis of a stable DNA copy of PSTV. Furthermore, RNA complementary to PSTV was not detected in infected tissues. Abbreviations: PSTV, potato spindle tuber viroid; Cot, initial concentration of DNA (mol of nucleotide per liter) X time (sec); Cort, initial concentration of RNA (mol of nucleotide per liter) X time (see); tm, temperature at which 50% of the DNA-RNA or RNA-RNA hybrid

denatures; SSC, standard saline-citrate solution (0.15 M sodium chloride-0.015 M sodium citrate, pH 7); X SSC means that the concentration of the solution used is times that of the standard saline-citrate solution. * This is paper XV in a series. Paper XIV is ref. 9. § A. Hadidi, M. J. Modak, and T. 0. Diener, in preparation.

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MATERIALS AND METHODS Preparation of Potato Spindle Tuber Viroid. A severe strain of PSTV analyzed here was originally isolated from potato plants and subsequently cultured and maintained in tomato plants for several years (10). PSTV was purified from tissues of PSTV-infected tomatoes as described (1, 11) and was labeled with 1251 in vitro (12). Specific activity of 125I-labeled PSTV was 105 cpm/ng of PSTV. Four different preparations of 1251-labeled PSTV prepared by W. Prensky, were used in this investigation. The 125I-labeled PSTV was assayed by fingerprinting and found to be at least 90% pure (13). Although iodinization leads to some random breakdown of RNA, iodinated PSTV was shown to contain a major component migrating at the position of the intact speciesin 10% polyacrylamide gels

(13).

Infection of Tomato or Gynura Plants with PSTV. Cotyledons of tomato seedlings or stems of young Gynura plants were inoculated mechanically, by rubbing or razor-slashing techniques, respectively, with partially purified preparations of PSTV. Plants were kept under light and temperature conditions favorable for PSTV synthesis. The newly developed young leaves were systemically infected with viroid. Systemic infection in plants, in contrast to local infection, implies that the majority of cells are infected. The viroid syndrome started to appear 2-3 weeks after inoculation; systemically infected leaves were harvested 2-5 days later and were kept at -80° until used for nucleic acid extraction. Preparation of Nucleic Acids. Nucleic acids were extracted from plant leaves as described by Diener (14) or Morris and Wright (15). DNA was spooled out on glass rods after addition of 95% ethanol and suspended in 0.01 M Tris-HCI buffer at pH 7.2. The DNA was sonicated and incubated in 0.3 M NaOH for 20-24 hr at 370 to hydrolyze RNA contaminants and to denature DNA (16). NaOH was removed by dialysis and DNA, concentrated by ethanol precipitation, was stored at -200 at afbut 10 mg/ml in H20 until used.for hybridization studies. DNA was isolated from several plant species. The common and generic names and family of each plant species are: tomato (Lycopersicon esculentum Mill. cv. Rutgers, Solanaceae); potato (Solanum tuberosum L., Solanaceae); cape gooseberry (Physalis peruviana L., Solanaceae); bean (Phaseolus vulgaris L., cv. Black Valentine, Leguminosae); Chinese cabbage (Brassica pekinensis Rupr., Cruciferae); purple passion (Gynura aurantiaca DC, Compositae); barley (Hordeum vulgare L., Gramineae). Total RNA from PSTV-infected or uninfected tomato was also extracted and tested for its complementarity to PSrV. Total RNA, recovered (after removing most of the DNA from the nucleic acid mixtures) by precipitation from ethanol (as described above), was treated with DNase (20 ,g/ml) in the presence of 3 mM MgCl2 at 370 for 1 hr. and then treated with

Proc. Nati. Acad. Sci. USA 73 (1976)

Cell Biology: Hadidi et al.

2454

i0

401

.1

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.r .

W

20

0.01 10

I&°

104 103 102 Cot (DNA) FIG. 1. Kinetics of hybridization of 125I-labeled PSTV with 0lo

various plant DNAs. Hybridization conditions were as described in Materials and Methods. Sources of DNA: 0-0-0, tomato; 0-0-0, PSTV-infected tomato; 0-0-0, potato; A-A--, bean; +-+-+, Gynura; *-*-*, PSTV-infected Gynura; and X-X-X, barley.

Pronase (300 14g/ml) at 370 for 3 hr. RNA was re-extracted with water-saturated phenol, precipitated with 95% ethanol, and dissolved in water. RNA precipitation from ethanol was repeated three more times. The RNA was finally dissolved in water and stored at -200 until used for hybridization. Samples of RNA preparations were analyzed in 2.4% polyacrylamide gels for 45 min. After electrophoresis, gels were scanned at 260 nm in a Gilford model 2400 recording spectrophotometer equipped with a linear transport systemT. The position of each RNA species in the electropherogram was typical of undegraded tomato RNA. PS'TVDNA Hybridization. 25I-Labeled PSTV (0.02 ng) was incubated with 50 ,Ag of fragmented cell DNA at 600 in 0.4 M sodium phosphate buffer at pH 6.8, as described earlier (16). Hybrids were scored by resistance to RNase in 3 X SSC (standard saline-citrate solution; 0.15 M sodium chloride-0.015 M sodium citrate, pH 7) at 370, for 60 min (16). The trichloroacetic acid-precipitable material was collected on Millipore filters and radioactivity was measured in a gamma counter. Hybridization values are expressed as the percentage of 125I-labeled PSTV recovered as RNase-resistant, trichloroacetic acid-precipitable radioactive material. This value is referred to here as "hybrid yield." PSTV*RNA Hybridization. 125I-Labeled PSTV (0.02 ng) was incubated at 600 with varying amounts of cellular RNA in 0.4 M sodium phosphate buffer at pH 6.8 (16). Some hybridization mixtures were boiled or heated at 1200 for 5 min before incubation at 600. Hybrid yield was assayed by determining resistance to RNase as described above. RESULTS Hybridization of PSTV to plant cell DNA To determine complementarity between PSTV and plant cell DNA, 125I-labeled PSTV was hybridized to DNA from PSTV-infected or uninfected tomato, PSTV-infected or uninfected Gynura, and uninfected potato, bean, or barley. The last ¶ Mention of a commercial company or specific equipment does not constitute its endorsement by the U.S. Government over similar equipment or companies not named.

RDNA

(sg=1)

FIG. 2. Double reciprocal plot from hybridization of a constant amount of 1251-labeled PSTV with an increasing amount of cellular DNA of phylogenetically diverse plants at a Cot value of 2 X 104. Hybrid formation and detection were done as described in Materials and Methods. Sources of DNA: 0-0-0, tomato; *-@--, PSTV-infected tomato; 0-0-0, potato; a-^-^, Physalis; A-A-A, bean; ++-+, Gynura; *-*-*, PSTV-infected Gynura; *-U--, Chinese cabbage; and X-X-X, barley.

two plant species are not known to be hosts of PSTV. The kinetics of hybridization in a typical experiment are shown in Fig. 1. With all DNAs, the hybrid yield increased gradually with time. At a Cot [initial concentration of DNA (mol of nucleotide per liter) X time (sec)] of 2 X 1i4 (336 hr), 35% of PSTV had hybridized to DNA from tissues of normal potatoes or tomatoes or to DNA from tissues of PSTV-infected tomatoes, and at this time the hybridization reaction had not yet reached completion. The nuclear DNA content of tomato is 3.9 pg per diploid cell (17). The calculated mass of tomato DNA per diploid cell is 2.35 X 1012 daltons. The genome size of tomatoes is therefore about % that of mammals. The Cot1/2 [' value of the initial concentration of DNA (mol of nucleotide per liter) X time (sec)] for maximum hybridization of reannealing of unique sequences in tomato DNA would be expected to be 1 to 3 X 103 and we expect the Cot1/2 of hybridization of 125I-labeled PSTV to unique sequences in tomato DNA to be around 5 to 10 X i@P. The observed COt1/2 of 6 X iOP for 125I-labeled PSTV hybridized to tomato DNA (Fig. 1) indicates that PSTV hybridizes to infrequent, possibly single copy DNA sequences. Similar results were obtained when PSTV was hybridized to DNA from bean tissues except that the formation of PSTV-DNA hybrid was slower. Lower hybrid yields resulted when PSTV was hybridized with DNA from Gynura (20% of the 125I-labeled PSTV hybridized). Hybrid kinetics or hybrid yield was not significantly different if DNA came from uninfected or PSTV-infected Gynura. Hybridization of PSTV with barley DNA resulted in the lowest levels of hybrid formation in kinetics experiments among plant species tested. To estimate hybrid yield at infinite DNA/RNA ratios, a constant amount of 125I-labeled PSTV was hybridized to increasing amounts of DNA (DNA titration), the time of hybridization and concentration of DNA being held constant. Fig. 2 shows a double reciprocal representation of results obtained with DNA from several plant species. Extrapolation to infinite DNA input (1/DNA = 0) indicates that 60% of the PSTV molecule is represented by complementary sequences in DNA of uninfected tomato, potato, Physalis peruviana, Black Valentine bean, and PSTV-infected tomato. The slopes of the DNA titration curves generated with DNA from solanaceous plants


Cell Biology: Hadidi et al.

2455

Table 1. Relationship of hybrid yield and tm of hybrids formed between PSTV and cellular DNAs Breadth of thermal transition

CP

Source of cell DNA

E

% PSTV

tm (0C)

(0C)

48

73

16

46 46

74 74

16 15

47 40

NT '73

NT 16

24

69

26

26

70

26

16 15

NT 69

NT 27

hybridized

60

Tomato

PSTV-infected

40

20

-

25 50 60 70 80 90 100

25

50 60 70 80 90 100

temperature PC)

FIG. 3. (A) Thermal stability of hybrids formed between 125Ilabeled PSTV and DNA from uninfected and PSTV-infected host tissue. Hybridization mixtures, each containing 500 ,g of DNA and 5 ng of 125I-labeled PSTV in 0.4 M sodium phosphate buffer, were boiled in capillary tubes for 5 min, and were then incubated at 60° to a Cot value of 1 X 104. Hybridization mixtures were expelled into 0.1 X SSC (1 X SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and aliquots of equal volume were exposed for 5 min to increasing increments of temperature. The amount of hybrid was measured as described in Materials and Methods. 0-0-0, tomato DNA; 0-0-0, PSTV-infected tomato DNA; A-A-A, Gynura DNA; A--A, PSTV-infected Gynura DNA. (B) Thermal stability of hybrids formed between 125I-labeled PSTV and DNA or RNA of uninfected or PSTV-infected tomato measured in 1 X SSC. tm was measured as described in (A), except that hybridization mixtures were expelled into 1 x SSC. O-O-O, tomato DNA; *---, PSTV-infected tomato PSTV-infected tomato RNA. DNA; ,-,-A, tomato RNA; A-A-A,

which indicate that PSTV-related sequences equal frequencies in those plants. With DNA from Black Valentine bean, however, the slope of the DNA titration curve is steeper, which suggests that PSTV-related sequences occur at lower frequency in DNA of this bean cultivar than in the DNAs of the solanaceous plants tested. DNA from uninfected or PSTV-infected Gynura aurantiaca contains sequences complementary to 30% of the PSTV molecule. Infection of G. aurantiaca with PSTV did not result in the formation of detectable new PSTV-related DNA sequences (Fig. 2) nor was amplification of "endogenous" PSTV-related sequences detectable. DNA titration experiments with DNA from Chinese cabbage or barley showed that these plants contain DNA sequences related to only a small portion of PSTV. Thermal stability of hybrids formed between PSTV and cellular DNA was measured to determine the degree of complementarity in the hybrid structures. Fig. 3 shows denaturation profiles of hybrids formed between l25-Ilabeled PSTV and DNA from PSTV-infected or uninfected plants. A tm (temperature at which 50% of the DNA-RNA hydrids denature) of 73744 in 0.1 X SSC (Fig. 3A) and a tm of 92° in 1 X SSC (Fig. SB) was obtained for hybrids formed between '25I-labeled PSTV and DNA of uninfected or PSTV-infected tomatoes. In both cases, the hybrid consisted primarily of a homogeneous population of well matched duplexes, since they had a high tm with a sharp denaturation profile. The high tm values of duplexes could be due to the high G-C content of PSTV. 32PLabeled PSTV has 55% G+C (A. Hadidi, unpublished). It is worth noting that hybrids formed between 125I-labeled RNA of 50% G+C (rRNA) and DNA have a tm value measured in 1 X SSC, of 82-870 depending on conditions of hybrid formation (16). Hybrids formed between 1251-labeled RNA of 40-45% were identical occur at about

tomato Potato Physalis peruviana Bean Gynura aurantiaca PSTV-infected Gynura Chinese cabbage Barley

The conditions of hybrid formation and detection were as outlined in Materials and Methods. Hybrids were formed at a Cot of 2 x 104 (192 hr). Thermal stability of hybrids was measured in 0.1 x SSC as described in Fig. 3A. NT = not tested.

G+C (Ig G mRNA) and DNA have a tm value measured in 1 X SSC of 78-830 (D. H. Gillespie, unpublished). tm values similar to those of PSTV-tomato DNA hybrids were obtained when 1251-labeled PSTV was hybridized to DNA from potato or bean (Table 1). A tm of 69-70° in 0.1 X SSC was obtained for duplexes formed between 125I-labeled PSTV and DNA of uninfected or PSTV-infected Gynura or barley DNA. The denaturation profiles were less sharp than those obtained when 1251-labeled PSTV was hybridized to DNA from solanaceous plants indicating more heterogeneity among the hybrids formed. A correlation between hybrid yield and tm of PSTV*DNA hybrids was observed (Table 1). In general, hybrids obtained in higher yield had high tm values and sharp transition profiles. The lower yield hybrids had low tm values and broad transition profiles. Hybridization of PSTV to cell RNA Experiments were done to determine whether tomatoes contain RNA sequences complementary to PSTV. Titration experiments using 1251-labeled PSTV and increasing amounts of RNA from PSTV-infected or uninfected tomato were carried to a Cort [initial concentration of RNA (mol of nucleotide per liter) X time (sec)] of 103. Previous experiments had shown the hybridization to be kinetically complete at this time. In all tests, the hybrid yields were small and did not significantly increase when the ratio of PSTV-infected or uninfected tomato RNA to a constant amount of 1251-labeled PSTV was increased (Fig. 4). The percentage of hybridization in hybrids formed between '251-labeled PSTV and RNA of PSTV-infected tomato was consistently higher than that obtained with hybrids formed between 1251-labeled PSTV and RNA of normal tomato, but not high enough to account for cell RNA sequences complementary to a large portion of PSTV. Furthermore, hybrids formed between '25I-labeled PSTV and RNA from PSTV-infected or uninfected tomato melted over a broad range of temperature with a tm of 760 in 1 X SSC (Fig. 3B), which indicates a large degree of mismatched duplex formation. In contrast, the tm of hybrids formed between l25I-labeled PSTV and DNA of

24.56

Proc. Nati. Acad. Scd. USA 73 (1976)

Cell Biology: Hadidi et al. 0~~~~~~~~~~~~~~~~~~~~~~~

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YRNA (g-1') FIG. 4. Double reciprocal plot from hybridization of a constant amount of 125I-labeled PSTV with an increasing amount of cellular RNA from uninfected or PSTV-infected tomato at a Cort value of 103. 0-0-0, tomato RNA; *-@--, PSTV-infected tomato RNA.

PSTV-infected or uninfected tomato was 920 in 1 X SSC and the hybrid melted over a narrow range of temperature (Fig. 3B), which indicates the formation of a well matched duplex of high G-C content. DISCUSSION

Evidence has been presented which shows that infrequent sequences complementary to PSTV are present in the DNAs of several uninfected host species of PSTV. At least 60% of PSTV is represented by sequences in the DNA of several solanaceous

host species. The DNA of a nonsolanaceous host, Gynura aurantiaca, contains sequences related to about 30% of the PSTV

molecule; however, in this

case, mismatched hybrids were formed. The DNAs of Chinese cabbage and barley, plants not known to be hosts of PSTV, contain sequences related to only a small portion of PSTV. Surprisingly, the DNA of Black Valentine bean, which also is resistant to infection by PSTV, contains sequences complementary to a large portion of the PSTV molecule. The frequency of PSTV-related DNA sequences, however, appears to be much lower in DNA of beans than in DNA from the solanaceous hosts. In general, the more distant, phylogenetically, plant species are from solanaceous plants (18), the fewer PSTV-related sequences their DNAs contain and the more distant they are from PSTV. These results support the hypothesis that PSTV originated from genes in normal sola-

plants. Evidently, an analogy exists between PSTV, and presumably other viroids, and the endogenous (class 1) RNA tumor viruses, whose genomes are closely related to DNA sequences in their uninfected natural hosts (19). To our knowledge, PSTV is the first infectious RNA agent of plants which possesses complementarity to its host DNA. Infection of tomato or Gynura plants with PSTV does not result in the appearance of detectable new sequences comple-

naceous

mentary to PSTV. These results are at variance with those in a recent report on citrus exocortis viroid (CEV), in which the authors claimed that 125I-labeled CEV hybridizes specifically with DNA-rich preparations from CEV-infected tomato or Gynura aurantiaca, but not with DNA-rich preparations from

uninfected tobacco, cowpea, tomato, or Gynura (20). The significance of this report, however, is uncertain because (i) hybridization reactions were made with RNA-DNA mixtures and not with purified DNA, (ii) the percentage of input CEV hy-

bridized was small (3.5% and 2.9%, for DNA-rich preparations from CEV-infected tomato and Gynura, respectively), and (tit) neither Cot values for hybrid formation nor thermal denaturation properties of the hybrids were reported. We were not able to detect, in infected tissue, RNA complementary to a major portion of PSTV, possibly because of an excess of plus PSTV. We did detect a partial complementarity between PSTV and RNA from uninfected or infected tomatoes. This finding also is similar to the partial complementarity found between RNA of RNA tumor viruses and cellular RNA (21). Since no new DNA sequences related to PSTV were found as a consequence of infection with PSTV and since involvement of DNA in PSTV replication has been indicated (8,9), preexisting host cell DNA sequences may be utilized during PSTV replication. Possibly, the introduction of PSTV into cells could act as a

regulatory signal, derepressing PSTV-specifying se-

quences and leading to viroid replication and disease development. In Gynura, where no exact DNA sequences complementary to PSTV were found, our experiments should have revealed the formation of new DNA sequences complementary to PSTV upon infection, provided that a large fraction of Gynura cells used for DNA purification became infected. Since no new DNA sequences complementary to PSTV were found upon infection, it is difficult to hypothesize that PSTV could be synthesized by the postulated derepression mechanism. The viroid released by PSTV-infected Gynura, however, has not, so far, been analyzed and it is possible that its sequence reflects the endogenous PSTV-related or even unrelated DNA sequences in Gynura, rather than the input PSTV molecule. We thank Dr. W. Prensky for labeling PSTV with 125I, Nancy P. Willingham for able technical assistance, and Muriel J. O'Brien for supplying Physalis peruviana and potato plants. We also thank Dr. R. C. Gallo for the use of facilities in his laboratory.

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1045-

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